Claus-Werner Franzke

Transmembrane collagen XVII, an epithelial adhesion protein, is shed from the cell surface by ADAMs

Collagen XVII, a type II transmembrane protein and epithelial adhesion molecule, can be proteolytically shed from the cell surface to generate a soluble collagen. Here we investigated the release of the ectodomain and identified the enzymes involved. After surface biotinylation of keratinocytes, the ectodomain was detectable in the medium within minutes and remained stable for >48 h. Shedding was enhanced by phorbol esters and inhibited by metalloprotease inhibitors, including hydroxamates and TIMP‐3, but not by inhibitors of other protease classes or by TIMP‐2. This profile implicated MMPs or ADAMs as candidate sheddases. MMP‐2, MMP‐9 and MT1‐MMP were excluded, but TACE, ADAM‐10 and ADAM‐9 were shown to be expressed in keratinocytes and to be actively involved. Transfection with cDNAs for the three ADAMs resulted in increased shedding and, vice versa, in TACE‐deficient cells shedding was significantly reduced, indicating that transmembrane collagen XVII represents a novel class of substrates for ADAMs. Functionally, release of the ectodomain of collagen XVII from the cell surface was associated with altered keratinocyte motility in vitro.

Complement Activation Triggers Metalloproteinases Release Inducing Cervical Remodeling and Preterm Birth in Mice

Inflammation is frequently linked to preterm delivery (PTD). Here, we tested the hypothesis that complement activation plays a role in cervical remodeling and PTD. We studied two mouse models of inflammation-induced PTD. The first model was induced by vaginal administration of lipopolysaccharide (LPS) and the second one by administration of progesterone antagonist RU486. Increased cervical C3 deposition and macrophages infiltration and increased serum C3adesArg and C5adesArg levels were observed in both models when compared to gestational age matched controls. A significant increase in collagen degradation, matrix metalloproteinase 9 (MMP-9) activity and tissue distensibility was observed in the cervix in both models. Mice deficient in complement receptor C5a did not show increased MMP-9 activity and cervical remodeling and did not deliver preterm in response to LPS or RU486, suggesting a role for C5aR in the cervical changes that precede PTD. In vitro studies show that macrophages release MMP-9 in response to C5a. Progesterone diminished the amount of C5aR on the macrophages surface, inhibited the release of MMP-9 and prevented PTD. In addition, macrophages depletion also prevented cervical remodeling and PTD in LPS-treated mice. Our studies show that C5a-C5aR interaction is required for MMP-9 release from macrophages, and the cervical remodeling that leads to PTD. Complement inhibition and supplementation with progesterone may be good therapeutic options to prevent this serious pregnancy complication.

Shedding of collagen XVII/BP180 in skin depends on both ADAM10 and ADAM9.

Collagen XVII is a transmembrane collagen and the major autoantigen of the autoimmune skin blistering disease bullous pemphigoid. Collagen XVII is proteolytically released from the membrane, and the pathogenic epitope harbors the cleavage site for its ectodomain shedding, suggesting that proteolysis has an important role in regulating the function of collagen XVII in skin homeostasis. Previous studies identified ADAMs 9, 10, and 17 as candidate collagen XVII sheddases and suggested that ADAM17 is a major sheddase. Here we show that ADAM17 only indirectly affects collagen XVII shedding and that ADAMs 9 and 10 are the most prominent collagen XVII sheddases in primary keratinocytes because (a) collagen XVII shedding was not stimulated by phorbol esters, known activators of ADAM17, (b) constitutive and calcium influx-stimulated shedding was sensitive to the ADAM10-selective inhibitor GI254023X and was strongly reduced in Adam10−/− cells, (c) there was a 55% decrease in constitutive collagen XVII ectodomain shedding from Adam9−/− keratinocytes, and (d) H2O2 enhanced ADAM9 expression and stimulated collagen XVII shedding in skin and keratinocytes of wild type mice but not of Adam9−/− mice. We conclude that ADAM9 and ADAM10 can both contribute to collagen XVII shedding in skin with an enhanced relative contribution of ADAM9 in the presence of reactive oxygen species. These results provide critical new insights into the identity and regulation of the major sheddases for collagen XVII in keratinocytes and skin and have implications for the treatment of blistering diseases of the skin.

Cleavage and oligomerization of gliomedin, a transmembrane collagen required for node of ranvier formation

Gliomedin, which has been implicated as a major player in genesis of the nodes of Ranvier, contains two collagenous domains and an olfactomedin-like domain and belongs to the group of type II transmembrane collagens that includes collagens XIII and XVII and ectodysplasin A. One characteristic of this protein family is that constituent proteins can exist in both transmembrane and soluble forms. Recently, gliomedin expressed at the tips of Schwann cell microvilli was found to bind axonal adhesion molecules neurofascin and NrCAM in interactions essential for Na+-channel clustering at the nodes of Ranvier in myelinating peripheral nerves. Interestingly, exogenously added olfactomedin domain was found to have the same effect as intact gliomedin. Here we analyze the tissue form of gliomedin and demonstrate that the molecule not only exists as full-length gliomedin but also as a soluble form shed from the cell surface in a furin-dependent manner. In addition, gliomedin can be further proteolytically processed by bone morphogenetic protein 1/Tolloid-like enzymes, resulting in release of the olfactomedin domain from the collagen domains. Interestingly, the later cleavage induces formation of higher order, insoluble molecular aggregates that may play important roles in Na+-channel clustering.

Shedding of Collagen XVII Ectodomain Depends on Plasma Membrane Microenvironment

Collagen XVII, a hemidesmosomal component, mediates the adhesion of epidermal keratinocytes to the underlying basement membrane. It exists as a full-length transmembrane protein and a soluble ectodomain that is proteolytically released from the cell surface by sheddases of a disintegrin and metalloproteinase (ADAM) family; TACE, the tumor necrosis factor-α-converting enzyme, is the major physiological proteinase. Because both collagen XVII and the ADAMs are transmembrane proteins, their plasma membrane microenvironment can influence shedding. Lipid rafts, assemblies of sphingolipids and cholesterol within the plasma membrane, are responsible for the separation of membrane proteins and are thought to regulate shedding of cell surface proteins. In this study we analyzed the influence of the cholesterol-depleting agent methyl-β-cyclodextrin (MβCD), which disintegrates lipid rafts, on the shedding of collagen XVII in HaCaT keratinocytes and in transfected COS-7 cells. Increasing concentrations of MβCD led to a dose-dependent decrease of membrane cholesterol levels and to stimulation of collagen XVII shedding. The stimulation was completely inhibited by sheddase inhibitors, and experiments with COS-7 cells co-transfected with TACE and collagen XVII demonstrated that TACE mediated the low cholesterol-dependent shedding. Co-patching analysis by double immunofluorescence staining revealed co-localization of collagen XVII with the raft resident phosphatidylinositol-linked placental alkaline phosphatase and segregation from the non-raft protein human transferrin receptor, indicating that a majority of collagen XVII molecules was incorporated into lipid rafts. These data deliver the first evidence for the role of plasma membrane lipid organization in the regulation of collagen XVII shedding and, therefore, in the regulation of keratinocyte migration and differentiation.

Ectodomain Shedding Generates Neoepitopes on Collagen XVII, the Major Autoantigen for Bullous Pemphigoid

As a type II transmembrane protein in basal keratinocytes, collagen XVII provides stable adhesion between epidermis and dermis in the skin. Its ectodomain can be shed from the cell surface, and autoantibodies in certain blistering diseases preferentially recognize the shed form. Major epitopes of collagen XVII are clustered within the juxtamembranous noncollagenous 16th A domain, and ectodomain shedding occurs within this region, suggesting that cleavage generates neoepitopes. However, the candidate cleavage sites have been controversial, and the mechanism of neoepitope generation is unclear. In this study, we investigated cleavage sites in the noncollagenous 16th A domain to understand the generation of neoepitopes and their pathological role. Polyclonal Abs recognizing the stretch Leu524-Gly532 preferentially reacted with the shed ectodomain, but not with the full-length form, indicating that a neoepitope was localized at this site. The neoepitope-specific Ab fixed complement and induced granulocyte-dependent dermal-epidermal separation in cryosections of normal human skin. The physiological cleavage sites were identified using mass spectrometry. N termini were found at Asp514, Leu524, Glu525, and Gly526, among which Asp514 and Glu525 were blocked by acetylation and pyroglutaminate. In silico prediction of B cell epitopes indicated that the antigenicity of the Leu524-Gly532 region increased substantially after shedding, regardless of the cleavage sites. Correspondingly, neoepitopes were found in the skin and blister fluids of patients with bullous pemphigoid, and bullous pemphigoid sera reacted with the peptide Leu524-Gly532. Taken together, these data demonstrate that physiological shedding of collagen XVII generates neoepitopes, which may serve as a target of blister-inducing autoantibodies.

Acral Peeling Skin Syndrome with TGM5 Gene Mutations May Resemble Epidermolysis Bullosa Simplex in Young Individuals

TO THE EDITOR The acral peeling skin syndrome (APSS) is a rare autosomal recessive condition characterized by superficial painless peeling of the skin predominantly on the dorsal aspects of hands and feet (Shwayder et al., 1997; Cassidy et al., 2005). The condition is usually aggravated by heat, humidity, and exposure to water. Microscopically, the cleavage level is located in the upper epidermis, between the stratum granulosum and the stratum corneum (Garcia et al., 2005). Only 15 patients with APSS have been reported since 1997 (Shwayder et al., 1997; Brusasco et al., 1998; Hashimoto et al., 2000; Cassidy et al., 2005; Garcia et al., 2005; Kharfi et al., 2009; Oumakhir et al., 2009; Wakade et al., 2009) (Table 1). In several of them, in addition to superficial peeling, acral blisters were also described (Wakade et al., 2009). The genetic basis of the disease was determined in only three families, in whom two different missense mutations in the TGM5 gene encoding transglutaminase 5 (TGase 5) were disclosed (Table 1) (Cassidy et al., 2005; Kharfi et al., 2009). It remains unclear whether the other patients have mutations in the same gene, or whether APSS is clinically and genetically heterogeneous (Cassidy et al., 2005). In this study, we investigated nine unrelated patients, eight children and one adult, clinically suspected to have epidermolysis bullosa simplex (EBS) because of acral skin blistering. The patients and/or diagnostic samples were referred to the Epidermolysis bullosa Center of the University Medical Center Freiburg (Volz et al., 2007) for molecular diagnostics of EBS. EDTA-blood and skin samples were obtained after informed consent of the patients and, if available, of family members. EDTAblood samples of 50 clinically unaffected Central European individuals were used as controls. The study was conducted according to the Declaration of Helsinki Principles. Immunofluorescence staining of skin cryosections was performed using a panel of antibodies to components of the epidermal basement membrane zone (Kern et al., 2006), as well as antibodies to loricrin (Abcam, Cambridge, UK), filaggrin (clone 15C10; Novocastra, Newcastle, UK), involucrin (clone SY5; Sigma, Taufkirchen, Germany), cytokeratin 10 (clone DE-K10; Dako, Glostrup, Denmark), TGase 1 (clone B.C1; Biomedical Technologies, Madrid Spain), TGase 3 (Jackson Immunoresearch Laboratories, West Grove, PA), and TGase 5 (Novus Biologicals, Littleton, CO). Genomic DNA was extracted from EDTA-blood using the QiAmp DNA mini kit (Qiagen, Hilden, Germany). Amplification of all KRT5 (NC_000012.11, National Center for Biotechnology Information (NCBI)), KRT14 (NC_000017.10, NCBI), and TGM5 (NC_000015.9, NCBI) exons and exon–intron boundaries, and sequencing were performed as described (Schuilenga-Hut et al., 2003; Wood et al., 2003; Cassidy et al., 2005). Mutations were confirmed by resequencing. The mutation c.763T4C was verified in 100 control chromosomes by Abbreviations: APSS, acral peeling skin syndrome; EBS, epidermolysis bullosa simplex; TGase, transglutaminase

Shedding of collagen XXIII is mediated by furin and depends on the plasma membrane microenvironment.

Collagen XXIII belongs to the class of type II orientated transmembrane collagens. A common feature of these proteins is the presence of two forms of the molecule: a membrane-bound form and a shed form. Here we demonstrate that, in mouse lung, collagen XXIII is found predominantly as the full-length form, whereas in brain, it is present mostly as the shed form, suggesting that shedding is tissue-specific and tissue-regulated. To analyze the shedding process of collagen XXIII, a cell culture model was established. Mutations introduced into two putative proprotein convertase cleavage sites showed that altering the second cleavage site inactivated much of the shedding. This supports the idea that furin, a major physiological protease, is predominantly responsible for shedding. Furthermore, our studies indicate that collagen XXIII is localized in lipid rafts in the plasma membrane and that ectodomain shedding is altered by a cholesterol-dependent mechanism. Moreover, newly synthesized collagen XXIII either is cleaved inside the Golgi/trans-Golgi network or reaches the cell surface, where it becomes protected from processing by being localized in lipid rafts. These mechanisms allow the cell to regulate the amounts of cell surface-bound and secreted collagen XXIII.

Transmembrane collagen XVII modulates integrin dependent keratinocyte migration via PI3K/Rac1 signaling.

The hemidesmosomal transmembrane component collagen XVII (ColXVII) plays an important role in the anchorage of the epidermis to the underlying basement membrane. However, this adhesion protein seems to be also involved in the regulation of keratinocyte migration, since its expression in these cells is strongly elevated during reepithelialization of acute wounds and in the invasive front of squamous cell carcinoma, while its absence in ColXVII-deficient keratinocytes leads to altered cell motility. Using a genetic model of murine Col17a1− /− keratinocytes we elucidated ColXVII mediated signaling pathways in cell adhesion and migration. Col17a1− /− keratinocytes exhibited increased spreading on laminin 332 and accelerated, but less directed cell motility. These effects were accompanied by increased expression of the integrin subunits β4 and β1. The migratory phenotype, as evidenced by formation of multiple unstable lamellipodia, was associated with enhanced phosphoinositide 3-kinase (PI3K) activity. Dissection of the signaling pathway uncovered enhanced phosphorylation of the β4 integrin subunit and the focal adhesion kinase (FAK) as activators of PI3K. This resulted in elevated Rac1 activity as a downstream consequence. These results provide mechanistic evidence that ColXVII coordinates keratinocyte adhesion and directed motility by interfering integrin dependent PI3K activation and by stabilizing lamellipodia at the leading edge of reepithelializing wounds and in invasive squamous cell carcinoma.

C-terminal truncation impairs glycosylation of transmembrane collagen XVII and leads to intracellular accumulation.

Collagen XVII, a type II transmembrane protein in hemidesmosomes, is involved in the anchorage of stratified epithelia to the underlying mesenchyme. Its functions are regulated by ectodomain shedding, and its genetic defects lead to epidermal detachment in junctional epidermolysis bullosa (JEB), a heritable skin fragility syndrome, but the molecular disease mechanisms remain elusive. Here we used a spontaneously occurring homozygous COL17A1 deletion mutant in JEB to discern glycosylation of collagen XVII. The mutation truncated the distal ectodomain and positioned the only N-glycosylation site 34 amino acids from the newly formed C terminus, which impaired efficient N-glycosylation. Immunofluorescence staining of authentic JEB keratinocytes and of COS-7 cells transfected with the mutant indicated intracellular accumulation of collagen XVII precursor molecules. Cell surface biotinylation and quantification of ectodomain shedding demonstrated that only about 15% of the truncated collagen XVII reached the cell surface. The cell surface-associated molecules were N-glycosylated in a normal manner, in contrast to the molecules retained within the cells, indicating that N-glycosylation of the ectodomain is required for targeting of collagen XVII to the plasma membrane and that reduced accessibility of the N-glycosylation site negatively regulates this process. Functional consequences of the strong reduction of collagen XVII on the cell surface included scattered deposition of cell adhesion molecule laminin 5 into the extracellular environment and, as a consequence of faulty collagen XVII-laminin ligand interactions, aberrant motility of the mutant cells.